Cellular mediators of innate immunity have the capacity to recognize and respond to target cells in the absence of antibody or T cells. Recently an ancient family of pathogen recognition receptors, the toll like receptors (TLRs) was discovered in humans and mice and several have been shown to trigger inflammatory responses to pathogen derived substances. We and others have shown that immature dendritic cells (iDC) express TLRs, and that TLRs on iDC drive maturation in response to pathogens, an essential component of acquired immunity. The TLR-driven maturation is modulated by G-protein coupled receptors, and we have also shown that the H2 histamine receptor, in particular, can profoundly affect the maturation process. Histamine is produced by mast cells, which are located in the same microenvironment as DC, and we demonstrated that human monocyte-derived iDC express two active histamine receptors, H1 and H2. Although histamine failed to affect the LPS-driven maturation of iDC with regard to phenotypic changes or capacity to prime naive T cells, it dramatically altered the repertoire of cytokines and chemokines secreted by mature DC. In particular, histamine, acting upon the H2 receptor for a short period of time, increased IL-10 production and reduced IL-12 secretion. As a result, histamine-matured DC polarized naive CD4+ T cells toward a Th2 phenotype, as compared with DC that had matured in the absence of histamine. We have also demonstrated the plasmacytoid DC respond to histamine by decreasing type-I interferon production, which could explain increased viral infections observed in some atopic patients. We are currently testing in mice the hypothesis that maturation of DC in a microenvironment that is rich in histamine will promote the generation of Th2 cells in the draining lymph nodes, thus favoring IgE production and leading to increased histamine secretion by mast cells. The TLR4 response to LPS requires the binding of MD-2 to its extracellular domain. MD-2 contains a leader sequence but lacks a transmembrane domain, and we asked whether it is secreted into the medium as an active protein. As a source of secreted MD-2 (sMD-2) we used culture supernatants from cells stably transduced with epitope-tagged human MD-2. We showed that sMD-2 exists as a heterogeneous collection of large disulfide linked oligomers formed from stable dimeric subunits, and that concentrations of sMD-2 as low as 50 pM enhanced the responsiveness of TLR4 reporter cells to LPS. An MD-2 like activity was also released by monocyte-derived iDC from normal donors. When co-expressed with MD-2, TLR4 indiscriminantly associated in the ER/cis Golgi with different sized oligomers of MD-2, and excess MD-2 was secreted into the medium. We concluded that normal and transfected cells secrete a soluble form of MD-2 that binds with high affinity to TLR4. Thus, sMD-2 might play a role in regulating responses to LPS and other pathogen derived substances in vivo. MD-2 contains seven Cys residues and to investigate the role of sulfhydryls in oligomerization and LPS responsiveness we created 22 single and multiple Cys to Ser mutants. All of the MD-2 mutants, including one totally lacking Cys residues, were secreted as mature, soluble and stable proteins. All were inactive when added as soluble protein to TLR4-expressing cells, but several mutants showed activity when co-transfected with TLR4. No single Cys residue was necessary for oligomerization and several were capable of forming either intra- or inter-chain disulfide bonds. We conclude that MD-2 is an atypical protein in which most if not all sulfhydryls are located on the surface of the molecule. Moreover, MD-2 is stable in the absence of sulfhydryls, suggesting that intrachain disulfide bonds do not play an essential role in tertiary structure formation, but may stabilize local structural features that are required for MD-2 function.